>Magister Scientiae - MSc / Approximately 50% of sorghum (Sorghum bicolour (L.) Moench) produced globally is used as human food, with 95% of its total consumption occurring in Africa. Unfortunately, sorghum crops are prone to pathogenic attack, notably leading to a reduction in production yields. Generally, chemical agents are used as fertilizers and/or biocides to increase crop production. However, these chemicals can have a detrimental environmental impact including the eutrophication of fresh water and marine ecosystems. Thus, there is increased interest in plant growth promoting rhizobacteria (PGPR), as an alternative to chemicals, to facilitate eco-friendly biological control of soil-borne pathogens. PGPRs colonize the plant root system (i.e rhizosphere and rhizoplane) and promote growth and production yields essentially via the biological control of plant pathogens and their role in the nutrient cycles (e.g N fixation). The aim of this study is to characterize the microbial communities associated with sorghum in South Africa, and to identify common bacteria which could further be developed and applied to improve sorghum growth and yield. Sorghum rhizospheric environments (rhizoplane and rhizosphere) were collected from three sites characterized by different agricultural practices (Free State, Limpopo and North West). Denaturing gradient gel electrophoresis (DGGE) and Terminal-restriction fragment length polymorphism (T-RFLP) were used to identify microbial community molecular fingerprints. Sorghum-associated microbial communities were found to be different in all rhizospheric soil samples which could be explained by differences in soil chemistry, agricultural practices and geographical location. The analyses also clearly demonstrated that the sorghum bacterial community structures were similar in the rhizoplane, indicating the strong influence that the sorghum plant has in determining the rhizoplane colonizers. The archaeal community structure from rhizoplane and rhizosphere in each sampling site were dissimilar, which could be explained by differences in soil type and/or agricultural practices. Both the T-RFLP and DGGE analyses revealed that Bacillus sp. were consistently associated with South African Sorghum, Arthrobacter sp. were detected in the rhizoplane, while Uncultured archaea were detected in the rhizoplane of sorghum. These microorganisms represent valuable targets for engineering to promote growth and yield in sorghum.

Soil structural stability plays a pivotal role in landscape preservation when a protective vegetation cover is lacking. For example, under semiarid climates seasonal rainfall cannot sustain a full vegetation cover, but still causes soil erosion. With the loss of (fertile) soil material, ecosystem productivity reduces and less C can be stored. In natural semiarid systems, soil erosion is a spatially heterogeneous process, whereby local highly erodible spots are alternated by improved soil structure under the sparse canopy cover, creating a very heterogeneous landscape. Although the physical protection by the plant canopy is well understood, the potential influence of soil archaea and bacteria on soil structural stability in relation to plants and parent material is less well known. Mainly from studies under controlled conditions, we know that certain archaeal and bacterial species have the ability to produce extracellular polymeric substances (EPS), forming an extracellular matrix. As the formed matrix connects soil particles, EPS seem to have the potential of playing a substantial role in soil aggregation, thereby controlling soil erodibility. Little is known of this gluing process by EPS and its importance under natural conditions as most evidence is derived from controlled conditions in the laboratory. This dissertation aims to unravel the role of EPS from soil archaea and bacteria in soil aggregate stabilization in semiarid grasslands by considering the potential role of plant species and parent material in this process. The sparse vegetation in semiarid grasslands provide a useful gradient in soil organic C contents to study these processes. Improved conditions for soil microbes producing EPS can be found at the root surface, while the bare canopy interspaces lack in C/resources. Two sites were selected in southeast Spain, mainly differing in graphitic C, inorganic C and nitrogen contents. On both sites, soil adjacent to the widely occurring Anthyllis cytisoides legumes shrubs and Macrochloa tenacissima grass tussocks were sampled during two campaigns. The first sampling campaign in April 2017 focused on the top soil, whereby a distance gradient from the plant stem to the bare intercanopy area was sampled. The second sampling campaign in April 2018 focused more on the effect of plant roots on soil archaeal and bacterial communities by including the rhizosphere. As the parent material of the Rambla Honda site, i.e. one of the study sites, contains a substantial amount of graphitic C, several methods were tested to quantify the different types of C in these soils to understand their role in shaping EPS contents. Furthermore, the quantification of graphitic C contents opened the possibility to study a potential interaction between graphite minerals and microbes. Although graphitic C contents explained part of the variances in microbial community, no direct link with EPS-saccharide contents was found. EPS contents were relative high in the rhizosphere, most notable at the legumes shrub Anthyllis cytisoides, and were linked to the enrichment of N-fixing bacteria. However, outside the root influenced soil, EPS contents were still substantially high, whereby the abundance of microbial species, previously associated to biofilm formation in other environments, indicated that EPS synthesis is not only restricted to the rhizosphere. Soil aggregation was linked to EPS-saccharide contents, whereby two mechanisms were hypothesized. Firstly, the strong link between soil wettability and EPS-saccharide content in the soil of the carbonate poor Rambla Honda site, indicated that aggregates become stabilized by hydrophobic bonds created by the EPS. Secondly, results from the carbonate rich Alboloduy site indicates that EPS has a facilitating role in creating stable aggregates by precipitating carbonates on the EPS structure. This likely lead to a higher soil structural stability, as carbonate bindings are more stable when prolonged drought reduces soil biological activity and thereby EPS contents. Overall, EPS play a substantial role in soil aggregate stabilization in semiarid grasslands, whereby EPS contents were increased by legume plants, by means of enriching EPS producing bacteria. / Die Stabilität der Bodenstruktur spielt eine entscheidende Rolle in der Erhaltung der Landschaft, insbesondere wenn keine schützende Vegetationsbedeckung vorhanden ist. So ist beispielsweise unter semiariden Klimabedingungen wegen der Saisonalität der Niederschläge keine vollständige Vegetationsbedeckung vorhanden, was Bodenerosion verursacht. Durch den Verlust von (fruchtbarem) Bodenmaterial verringert sich die Produktivität des Ökosystems. Dadurch kann weniger Kohlenstoff (C) im Boden gespeichert werden. In natürlichen semiariden Systemen ist die Bodenerosion ein räumlich heterogener Prozess, bei dem sich stark erosionsanfällige Stellen mit solchen Bereichen abwechseln, welche durch günstige Bodenstruktur unter der spärlichen Pflanzendecke gekennzeichnet sind. Hierdurch entsteht eine sehr heterogene Landschaft. Während zum physikalischen Schutz durch Vegetationsüberschirmung viele Erkenntnisse vorliegen, ist über den möglichen Einfluss von Archaeen und Bakterien auf die strukturelle Stabilität des Bodens in Bezug auf Pflanzen und Ausgangsmaterial weit weniger bekannt. Hauptsächlich aus Studien unter kontrollierten Bedingungen wissen wir, dass bestimmte Archaen- und Bakterienarten die Fähigkeit besitzen, extrazelluläre polymere Substanzen (EPS) zu produzieren, die eine extrazelluläre Matrix bilden. Da die gebildete Matrix Bodenpartikel verbindet, scheint EPS das Potenzial für eine maßgebliche Beeinflussung der Bodenaggregation zu haben und dadurch die Erosionsanfälligkeit zu steuern. Über solche Klebemechanismen von EPS und deren Bedeutung unter natürlichen Bedingungen ist aber wenig bekannt; die meisten Hinweise stammen aus kontrollierten Bedingungen im Labor. Diese Dissertation zielt darauf ab, die Bedeutung von EPS von Archaeen und Bakterien hinsichtlich der Stabilisierung von Bodenaggregaten in semiariden Graslandschaften unter Berücksichtigung der möglichen Rolle von Pflanzenarten und Ausgangsmaterial in diesem Prozess aufzuklären. Zur Untersuchung solcher Prozesse bietet die spärliche Vegetation in semiariden Graslandschaften einen zweckdienlichen Gradienten bezüglich des Gehalt an organischem C im Boden. Günstige Bedingungen für EPS-produzierende Bodenmikroorganiosmen sind an der Wurzeloberfläche zu finden, während dem unbedeckten Boden zwischen Stellen ohne Pflanzenbedeckung C / Ressourcen fehlen. Es wurden zwei Standorte in Südostspanien ausgewählt, die sich hauptsächlich in den Gehalten an graphitischem C, anorganischem C und Stickstoff unterscheiden. An beiden Standorten wurden im Rahmen von zwei Feldkampagnen Böden in unmittelbarer Nähe zu der weit verbreiteten Leguminosenart Anthyllis cytisoides-Hülsenfrüchten und Grasbüscheln von Macrochloa tenacissima beprobt. Die erste Probenahmekampagne im April 2017 konzentrierte sich auf den obersten Boden, wobei ein Abstandsgradient vom Pflanzenspross zum unbedeckten Boden zwischen der Pflanzendecke beprobt wurde. Die zweite Probenahmekampagne im April 2018 konzentrierte sich mehr auf die Wirkung von Pflanzenwurzeln auf Archaeen- und Bakteriengemeinschaften durch Beprobung der Rhizosphäre. Am Rambla Honda-Standort enthält das Ausgangsmaterial eine erhebliche Menge an graphitischem C. Deshalb wurden verschiedene Methoden getestet, um die verschiedenen Arten von C in diesen Böden zu quantifizieren und ihre Rolle bei der Gestaltung des EPS-Gehalts zu verstehen. Darüber hinaus eröffnete die Quantifizierung des graphitischen C-Gehalts die Möglichkeit, die Wechselwirkung zwischen Graphitmineralen und Mikroorganismen zu untersuchen. Obwohl der Gehalt an graphitischem C einen Teil der Varianzen in der mikrobiellen Gemeinschaft erklärte, wurde kein direkter Zusammenhang mit dem EPS-Saccharidgehalt gefunden. Die EPS-Gehalte waren in der Rhizosphäre relativ hoch - am deutlichsten bei der Leguminosenart Anthyllis cytisoides - und mit der Anreicherung von N-fixierenden Bakterien verbunden. Außerhalb des von der Wurzel beeinflussten Bodens war der EPS-Gehalt jedoch immer noch deutlich erhöht. Dabei wies die Häufigkeit von Mikroorganismenarten, die zuvor mit der Bildung von Biofilmen in anderen Umgebungen in Verbindung gebracht wurden, darauf hin, dass die EPS-Synthese nicht nur auf die Rhizosphäre beschränkt ist. Die Bodenaggregation zeigte eine Verbindung mit dem EPS-Saccharidgehalt auf, wobei zwei Mechanismen angenommen wurden: Erstens wies der starke Zusammenhang zwischen der Bodenbenetzbarkeit und dem EPS-Saccharidgehalt im Boden des karbonatarmen Rambla Honda-Standorts auf eine Aggregatstabilisierung durch EPS-erzeugte hydrophobe Bindungen hin. Zweitens zeigen die Ergebnisse des Standorts Alboloduy-Standorts mit karbonatreichem Boden, dass EPS eine unterstützende Funktion bei der Erzeugung stabiler Aggregate besitzt, indem Karbonate auf der EPS-Struktur ausgefällt werden. Dies führt wahrscheinlich zu einer höheren Stabilität der Bodenstruktur, da Karbonatbindungen stabiler sind, wenn eine längere Trockenheit zu einer Verringerung der biologischen Aktivität im Boden und damit des EPS-Gehalts führt. Insgesamt spielt EPS eine wesentliche Rolle bei der Stabilisierung von Bodenaggregaten in semiariden Graslandschaften, wobei der EPS-Gehalt durch Leguminsosen, mittels Anreicherung von EPS-produzierenden Bakterien, erhöht wurde.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-29T13:49:53Z No. of bitstreams: 1 Andresa Priscila Souza Ramos.pdf: 3687585 bytes, checksum: 03a2a3149474293923ca08c0db68bc60 (MD5) / Made available in DSpace on 2016-06-29T13:49:53Z (GMT). No. of bitstreams: 1 Andresa Priscila Souza Ramos.pdf: 3687585 bytes, checksum: 03a2a3149474293923ca08c0db68bc60 (MD5) Previous issue date: 2011-08-26 / The association between bacteria and plants is considered extremely important because it may promote the plant development by providing nutrients, protection, and production of plant growth promoting substances like indole acetic acid (IAA). The sugarcane production is under a great economic and agricultural importance, and increasing its production still depending on several chemical inputs that are expensive and environmentally damaging. A sustainable alternative is the use of beneficial bacteria that may diminish or abolish the use of these inputs and increase agricultural production. Therefore, the objectives of this study were: i) selecting and evaluating IAA producing bacteria, dependent and independent pathway of tryptophan, sugarcane roots plant association in crop fields in Pernambuco, ii) evaluate the influence of niche isolation, the place of cultivation, the cultivation time and host plant genotype on frequency and IAA-producing bacteria; iii) Analyze the genetic variability of IAA-producing bacteria by BOX-PCR, and iv) assess the noncultivable bacterial community sugarcane ratoon-associated v) Evaluate the production of IAA, two endophytic bacteria according to the time. Isolates were studied at four and ten months sugarcane plant cultivation, collected in EECAC Carpina-PE and Petribú factory,Lagoa de Itaenga-PE and isolates from three months on sugarcane plant-ratoon regrowth, collected in EECAC. The results showed that plants from sugarcane crop fields in Pernambuco have a high frequency of bacteria producing IAA in vitro by a tryptophan-dependent and independent pathways, the crop place, cultivation time of the host plant and niche colonization. Bacterial influence on the distribution of IAA-producing bacteria. In addition, the location, time of cultivation of the host plant genotype and bacterial niche colonization influenced the production levels. The strains showed high genetic variability. In sugarcane plant-ratoon, the bacterial population density was higher than in the rizhosphericsoil and root-endophytic bacteria also showed a high frequency capacity to produce IAA in vitro. Analysis of variability and genetic diversity revealed high genetic variability among bacteria producing IAA and the predominance of dominant bacterial groups not grown in soil and inside the roots of sugarcane plant-ratoon grown in Pernambuco. Some strains of root endophytic sugarcane plant, with capacity to produce IAA, were identified as belonging to the genus Enterobacter and Burkholderia / A associação entre bactérias benéficas e plantas é considerada extremamente importante, pois pode promover o desenvolvimento do vegetal através da disponibilização de nutrientes, proteção e produção de substâncias promotoras de crescimento vegetal, como o ácido indol acético (AIA). A canade- açúcar é uma cultura de grande importância agrícola e econômica, e sua produção crescente ainda depende de diversos insumos químicos que são onerosos e prejudicam o ambiente. Uma alternativa sustentável seria a utilização de bactérias benéficas que possam diminuir ou anular o uso desses insumos e incrementar a produção agrícola. Diante disso, os objetivos deste trabalho foram: i) selecionar e avaliar bactérias produtoras de AIA, via dependente e independente de triptofano, associadas a raízes de plantas de cana-de-açúcar cultivadas em Pernambuco; ii) avaliar a influência dos nichos de colonização, do local e tempo de cultivo e do genótipo da planta hospedeira sobre a frequência e produção de bactérias produtoras de AIA; iii) analisar a variabilidade genética de bactérias produtoras de AIA por BOX-PCR; iv) avaliar a comunidade bacteriana não cultivável associada a cana soca; v) e avaliar a produção de AIA, de duas bactérias endofíticas, em função do tempo. Foram estudadas bactérias isoladas aos quatro e dez meses de cultivo de cana planta, coletadas na EECAC- Carpina-PE e na Usina Petribú- Lagoa de Itaenga-PE e linhagens isoladas aos três meses de rebrota na cana soca, coletadas na EECAC. Os resultados demonstraram que as plantas de cana-deaçúcar cultivadas em Pernambuco apresentam alta frequência de bactérias com capacidade de produzir AIA in vitro, via dependente e independente de triptofano, e que o local, tempo de cultivo da planta hospedeira e o nicho de colonização bacteriana influenciaram na distribuição das bactérias produtoras de AIA. Além disso, o local, tempo de cultivo, genótipo da planta hospedeira e o nicho de colonização bacteriana influenciaram nos níveis de produção e as linhagens apresentaram alta variabilidade genética. Na cana soca, a densidade populacional bacteriana foi maior no solo rizosférico do que endofítica de raiz e também apresentaram alta frequência de bactérias com capacidade de produzir AIA in vitro. A análise da variabilidade e diversidade genética revelaram alta variabilidade genética entre as bactérias produtoras de AIA e a predominância de grupos bacterianos dominantes, não cultivados, no solo e no interior das raízes de cana soca, cultivadas em Pernambuco. Algumas linhagens endofíticas de raiz de cana planta, com capacidade de produzir AIA, foram identificadas como pertencentes aos gêneros Burkholderia e Enterobacter